Diagnostic methods for cardiovascular disease, low hdl-cholesterol levels, and high triglyceride levels

ABSTRACT

A method for determining propensity toward developing a cardiovascular disease in a patient at risk thereof by determining the presence in an ABCA1 gene of said patient of a polymorphism in the DNA sequence of the gene wherein said polymorphism is present in a non-coding region of said gene is disclosed. Also described is a method of identifying a modulator of ABCA1 polynucleotide expression comprising by determining the ability of a test compound to modulate the activity of a polynucleotide comprising a polymorphism disclosed herein, or to mimic the effects of such polymorphism where such effects are beneficial. Treatment of cardiovascular disease, especially coronary artery disease, using agents identified by the disclosed methods is also described.

This application claims priority of U.S. Provisional Application60/293,742, filed 25 May 2001, the disclosure of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of gene polymorphisms,especially single nucleotide polymorphisms present in non-coding regionsof the ABCA1 gene, and their use in diagnosing risk of cardiovasculardisease, including coronary artery disease, and in screening forcompounds useful in mimicking advantageous polymorphisms and for agentsthat enhance the activity of beneficial polymorphisms.

BACKGROUND OF THE INVENTION

Atherosclerotic cardiovascular disease is the leading cause of deathworldwide¹. Altered lipoprotein levels are pivotal risk factors foratherosclerosis^(2,3). In particular, low HDL cholesterol (HDL-C) levelsare a major independent risk factor for the development of prematurecoronary disease⁴⁻⁶. The anti-atherogenic function of HDL is generallyattributed to its role in reverse cholesterol transport (RCT), wherebyexcess cholesterol is transported from peripheral cells to HDL particlesfor subsequent delivery to the liver^(7,8). The protein crucial for theinitial step of RCT, namely ABC1, was recently identified⁹⁻¹².

Complete ABC1 deficiency is the underlying cause of Tangier disease(TD)^(9,11,12), a rare disorder associated with a near absence of HDL-Cand apolipoprotein AI and with remarkably decreased cholesterol effluxfrom cells¹³. Clinically, TD is associated with hepatosplenomegaly,neuropathy and cholesterol ester accumulation in specific cells¹³.Individuals heterozygous for ABC1 mutations are characterized by lowHDL-C levels, increased triglycerides (TG), depressed levels ofcholesterol efflux and an increased risk of coronary artery disease(CAD), but have no obvious clinical manifestations of cholesterol esteraccumulation^(9,10,14). Cholesterol efflux levels are highly correlatedwith HDL-C levels in these individuals¹⁴. The frequency of individualswith severe mutations in the ABC1 gene is low, but common variantshaving minor functional effects could be of great clinical relevance forthe general population.

We have previously shown that individuals heterozygous for mutations inthe ABC1 gene (also called ABCA1) have decreased HDL cholesterol(HDL-C), increased triglycerides (TG) and a greater than threefoldincreased frequency of coronary artery disease (CAD) and that singlenucleotide polymorphisms in the coding region (cSNPs) of the ABC1 genemay significantly impact plasma lipid levels and the severity of CAD inthe general population. We have now identified several SNPs innon-coding regions of ABC1 that may be important for the appropriateregulation of ABC1 expression (i.e. in the promoter, intron 1 and the 5′untranslated region (UTR)), and have examined the phenotypic effects ofthese SNPs in the REGRESS population. Of 12 SNPs, 4 were associated witha clinical outcome. A 3-fold increase in coronary events and anincreased family history of CAD was evident for the G-191C variant.Similarly, the C69T SNP was also associated with a 2-fold increase inevents. In contrast, the C-17G was associated with decreased coronaryevents, and the InsG319 SNP was associated with less focal and diffuseatherosclerosis. For all these SNPs, the changes in atherosclerosis andCAD occurred independent of changes in plasma lipid levels, findingswhich were replicated in a second cohort. These data suggest that commonvariation in non-coding regions of ABC1 may significantly alter theseverity of atherosclerosis, without necessarily influencing plasmalipid levels.

We have previously presented a complete analysis of 10 single nucleotidepolymorphisms in the coding region of the ABC1 gene (cSNPs)¹⁵. We haveshown that cSNPs of the ABC1 gene influence plasma lipid levels and theseverity of CAD. Interestingly, the R219K cSNP is associated withdecreased TG, increased HDL-C and a decreased severity of CAD,compatible with a gain of function, while other cSNPs were associatedwith more moderate effects¹⁵.

Here, we describe 12 non-coding SNPs in potential regulatory regions andhave examined the functional effects of these SNPs in the promoter, the5′ untranslated region (UTR) and first intron. Several studies haveshown that SNPs in these regions from other genes indeed have functionalconsequences ¹⁶⁻¹⁸. We have also recently shown that sequences withinthe first intron of ABC1 constitute an alternate promoter with threealternate transcription start sites, and thus may have direct effects onthe regulation of ABC1 (Singaraja et al, manuscript submitted). Analternate transcription start site within intron 1 has also recentlybeen reported by another group¹⁹.

We have now examined the phenotypic effects of these 12 non-coding SNPsin a large ethnically uniform cohort (REGRESS) and show that they indeedare associated with altered risk and severity of CAD, without associatedchanges in lipid and lipoprotein levels. This provides evidence thatsequences in these regions are important for the proper regulation ofABC1 and suggest that changes in ABC1 regulation can alter risk for CADpresumably through influencing RCT without necessarily having an effecton lipid levels.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method for determiningpropensity toward developing a cardiovascular disease in a patient atrisk of developing said disease comprising determining the presence inan ABCA1 gene of said patient of a polymorphism in the DNA sequence ofsaid gene wherein said polymorphism is present in a non-coding region ofsaid gene.

In preferred embodiments, the polymorphism is present in the promoterregion of said gene or in an intronic region.

In preferred embodiments, the disease is coronary artery disease oratherosclerosis, or a disease that involves increased triglyceride orcholesterol levels, or decreased HDL-C levels, in a patient, especiallyin the plasma of said patient.

In preferred embodiments, the disease involves decreased lipid transportin the cells of the patient, especially decreased HDL-C transport.

In additional preferred embodiments, the polymorphism is a singlenucleotide polymorphism, most preferably any of the polymorphismsdepicted in Table 1 (SEQ ID NOS: 1-24).

In another aspect, the present invention relates to method foridentifying a modulator of ABCA1 polynucleotide expression comprising:

-   -   (a) contacting a compound with a polynucleotide that encodes        ABCA1 polypeptide, which polynucleotide comprises a polymorphism        in a non-coding region of said polynucleotide, under conditions        promoting said contacting and promoting expression of ABCA1        polypeptide by said polynucleotide;    -   (b) determining the activity of said polynucleotide in        expressing said ABCA1 polypeptide after said contacting wherein        a difference in the expression of said polynucleotide relative        to when said compound and said polynucleotide are not contacted        indicates polynucleotide modulating activity,    -   thereby identifying a modulator of ABCA1 polynucleotide        expression.

In a preferred embodiment, the ABCA1 polynucleotide is present in acell, which cell then expresses the ABCA1 polypeptide and suchexpression is readily measured, such as by measuring lipid transportacross the membrane of the cell whereby an increase in transport showsincreased expression of the polypeptide. Thus, in a preferredembodiment, the difference in expression in step (b) is an increase inexpression. Preferably, the polymorphism is present in an intronicregion or promoter region, or some other non-coding region, such as anenhancer region, of the polynucleotide.

In a preferred embodiment, the polymorphism is a single nucleotidepolymorphism (SNP), most preferably one of the SNPs shown in Table 1(SEQ ID NOS: 1-24).

Such polymorphisms may also have the effect of decreasing the activityof said polynucleotide.

In a further aspect, the present invention relates to a method foridentifying an agent that modulates plasma lipid levels comprisingadministering to an animal an effective amount of a compound firstidentified as an ABCA1 modulator using a screening method as disclosedherein. In preferred embodiments thereof, the compound has the effect ofreducing plasma triglyceride levels, reducing plasma cholesterol levels,or increasing plasma HDL-C levels.

In an additional aspect, the present invention relates to a method oftreating a patient for cardiovascular disease comprising administeringto a patient afflicted therewith of an effective amount of a compoundfirst identified as an ABCA1 modulator using a screening method asdisclosed herein. In preferred embodiments, the disease is coronaryartery disease or atherosclerosis.

In yet a further aspect, the present invention relates to a method ofprotecting a patient against developing cardiovascular diseasecomprising administering to a patient at risk thereof of an effectiveamount of a compound first identified as an ABCA1 modulator using themethod as disclosed herein. In preferred embodiments thereof, thedisease is coronary artery disease or atherosclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the location of non-coding SNPs in thepromoter, intron 1 and the 5′ untranslated regions of exons 1 and 2 ofthe ABC1 gene. The alternate exons we have recently identified withinintron 1 are also indicated (exons 1 b, c, d; Singaraja et al,manuscript submitted). The translation initiation (ATG) site in exon 2is indicated. SNPs that are in complete or near complete linkagedisequilibrium are joined by dashed lines. Variants marked by a * werepreviously reported in reference²¹. The diagram is not drawn to scale.

FIG. 2. Event-free survival by G-191C genotype. The curves represent thecumulative proportion of the cohort that was event-free during the trial(thin line for AA, thick line for AB and dashed line for BB). Homozygouscarriers of this variant (BB) had significantly more events during thetwo-year trial than individuals with either of the other genotypes.

FIG. 3. Event-free survival in C69T carriers and non-carriers. Thecurves (AB+BB− thick line, AA− thin line) represent the cumulativeproportion of the cohort that was event-free during the trial. Carriersof this variant had significantly more events during the two-year trialthan non-carriers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features diagnostically relevant polymorphisms ofthe human ABC1 gene regulatory region. In particular, we have determinedthe statistical relationship between certain SNPs in the regulatorydomain of the ABC1 gene and the incidence of coronary events andcoronary artery disease in humans. This relationship establishes theimportance and utility of diagnostic assays which identify the presenceor absence of such SNPs in a human. For example, identification of theseSNPs can have medical use in (1) diagnosis of disease and predictingdisease progression; (2) selection of drugs for patients based onimproved efficacy or reduced side-effects; and (3) selection of patientsfor enrolment in clinical trials and classification of patients inclinical trials by ABC1 genotype. These polymorphisms are also useful inany of the diagnostic assays involving ABC1 nucleic acids or proteinsthat are described in PCT Publication WO 00/55318, filed Mar. 15, 2000;U.S. Utility application Ser. No. 09/526,193, filed Mar. 15, 2000; PCTPublication WO 00/0115676, filed Sep. 1, 2000; or U.S. Utilityapplication Ser. No. 09/654,323, filed Sep. 1, 2000 (which are eachherein incorporated by reference). In addition, see Zwarts et al, ABCA1regulatory variants influence coronary artery disease independent ofeffects on plasma lipid levels, Clin. Genet. 61(2): 115-25 (February2002), the disclosure of which is hereby incorporated by reference inits entirety.

For example, determination of the genetic subtyping of ABC1 genesequences can be used to subtype individuals or families with lower thannormal HDL cholesterol levels or higher than normal triglyceride levelsto determine whether the lower than normal HDL or higher than normaltriglyceride phenotype is related to ABC1 function. This diagnosticprocess can lead to the tailoring of drug treatments according topatient genotype (referred to as pharmacogenomics), including predictionof the patient's response (e.g., increased or decreased efficacy orundesired side effects upon administration of a compound or drug). Thesediagnostic methods may also be used to determine a subject's risk for acardiovascular disease, such as coronary artery disease,atherosclerosis, myocardial infarction, ischemic attack, angina,peripheral vascular disease, or stroke.

In one such aspect, the invention features a method for predicting aperson's response to a drug by determining whether the person has apolymorphism in an ABC1 gene, promoter, or regulatory sequence thatalters the person's response to the drug. Examples of therapeutic agentsthat can be used in these methods include triglyceride-lowering drugs,HDL cholesterol-raising drugs, and agents for the treatment orprevention of cardiovascular disease, such as coronary artery disease.

In another aspect, the invention features a method of determining asubject's propensity for a disease or condition selected from the groupconsisting of a lower than normal HDL cholesterol level, a higher thannormal triglyceride level, and a cardiovascular disease. This methodinvolves determining the presence or absence of at least one ABC1polymorphism in the polynucleotide sequence of an ABC1 regulatoryregion, promoter, or coding sequence or in the amino acid sequence of anABC1 protein in a sample obtained from the subject, wherein the presenceor absence of the ABC1 polymorphism is indicative of a risk for thedisease or condition. Desirably, the method also includes analyzing atleast five ABC1 polymorphic sites in the polynucleotide sequence or theamino acid sequence.

In yet another aspect, the invention features a method for determiningwhether an ABC1 polymorphism is indicative of a risk in a subject for adisease or condition selected from the group consisting of a lower thannormal HDL cholesterol level, a higher than normal triglyceride level,and a cardiovascular disease. The method includes (a) determiningwhether the prevalence of the disease or condition in a first subject orset of subjects differs from the prevalence of the disease or conditionin a second subject or set of subjects; (b) analyzing the polynucleotidesequence of an ABC1 regulatory region, promoter, or coding sequence orthe amino acid sequence of an ABC1 protein in a sample obtained from thefirst subject or set of subjects and the second subject or set ofsubjects; and (c) determining whether at least one ABC1 polymorphismdiffers between the first subject or set of subjects and the secondsubject or set of subjects, wherein the presence or absence of the ABC1polymorphism is correlated with the prevalence of the disease orcondition, thereby determining whether the ABC1 polymorphism isindicative of the risk. Desirably, the method further includes analyzingat least five ABC1 polymorphic sites in the polynucleotide sequence ofan ABC1 regulatory region, promoter, or coding sequence or in the aminoacid sequence of ABC1.

In another aspect, the invention provides an electronic database havinga plurality of sequence records of ABC1 polymorphisms correlated torecords of predisposition to or prevalence of a disease or conditionselected from the group consisting of a lower than normal HDLcholesterol level, a higher than normal triglyceride level, and acardiovascular disease.

In another aspect, the invention features a method for selecting adesirable therapy for modulating ABC1 activity or expression in asubject. This method includes (a) determining the presence or absence ofat least one ABC1 polymorphism in the polynucleotide sequence of an ABC1regulatory region, promoter, or coding sequence or in the amino acidsequence of an ABC1 protein in a sample obtained from the subject,wherein the presence or absence of the ABC1 polymorphism is indicativeof the safety or efficacy of at least one therapy for modulating ABC1expression or activity; and (b) determining a desirable therapy formodulating ABC1 expression or activity in the subject. Desirably, themethod further includes analyzing at least five ABC1 polymorphic sitesin the polynucleotide sequence of an ABC1 regulatory region, promoter,or coding sequence or the amino acids sequence of ABC1.

The invention also includes methods, compositions, and kits which areuseful for identification of the herein disclosed SNPs in a subject(e.g., a human).

In embodiments of any of the various aspects of the invention, thepolymorphism is one or more of the polymorphisms listed in Table 1 ordescribed herein (SEQ ID NOS: 1-24). In other desirable embodiments, thepolymorphism is in the 5′ regulatory region of ABC1.

In accordance with the foregoing, the present invention relates to amethod for determining propensity toward developing a cardiovasculardisease in a patient at risk of developing said disease comprisingdetermining the presence in an ABCA1 gene of said patient of apolymorphism in the DNA sequence of said gene wherein said polymorphismis present in a non-coding region of said gene. As used therein, thepolymorphism is present in the promoter region of said gene or in anintronic region or some other non-coding region, as described in FIG. 1,especially where the polymorphism is a single nucleotide polymorphism.

The diseases to be diagnosed include any type of cardiovascular disease,such as, but in no way limited to, coronary artery disease oratherosclerosis, wherein the disease involves increased triglyceride orcholesterol levels, or decreased HDL-C levels, in a patient, especiallywherein the plasma levels of the patient reflect these increased ordecreased lipid levels. Such diseases also involve decreased lipidtransport in the cells of the patient, especially decreased HDL-Ctransport.

The present invention also contemplates a method for identifying amodulator of ABCA1 polynucleotide expression comprising:

-   -   (a) contacting a compound with a polynucleotide that encodes        ABCA1 polypeptide, which polynucleotide comprises a polymorphism        in a non-coding region of said polynucleotide, under conditions        promoting said contacting and promoting expression of ABCA1        polypeptide by said polynucleotide;    -   (b) determining the activity of said polynucleotide in        expressing said ABCA1 polypeptide after said contacting wherein        a difference in the expression of said polynucleotide relative        to when said compound and said polynucleotide are not contacted        indicates polynucleotide modulating activity,    -   thereby identifying a modulator of ABCA1 polynucleotide        expression.

As used in such methods, the ABCA1 polynucleotide may be present in acell, which cell then expresses the ABCA1 polypeptide and suchexpression is readily measured, such as by measuring lipid transportacross the membrane of the cell whereby an increase in transport showsincreased expression of the polypeptide. Thus, in a preferredembodiment, the difference in expression in step (b) is an increase inexpression. Preferably, the polymorphism is present in an intronicregion or promoter region, or some other non-coding region, such as anenhancer region, of the polynucleotide, especially where thepolymorphism is a single nucleotide polymorphism (SNP), most preferablyone of the SNPs shown in Table 1 (SEQ ID NOS: 1-24). Such polymorphismsmay also have the effect of decreasing the activity of saidpolynucleotide.

In accordance with the methods of the foregoing, the present inventionprovides a method for identifying an agent that modulates plasma lipidlevels comprising administering to an animal an effective amount of acompound first identified as an ABCA1 modulator using a screening methodas disclosed herein. In preferred embodiments thereof, the compound hasthe effect of reducing plasma triglyceride levels, reducing plasmacholesterol levels, or increasing plasma HDL-C levels.

Because such agents are useful in treating diseases of lipid metabolism,the present invention provides a method of treating a patient forcardiovascular disease comprising administering to a patient afflictedtherewith of an effective amount of a compound first identified as anABCA1 modulator using a screening method as disclosed herein. Inpreferred embodiments, the disease is coronary artery disease oratherosclerosis.

Agents identified according to the screening assays disclosed hereinalso find use in preventing lipid-related diseases from developing andthus the present invention provides methods of protecting a patientagainst developing cardiovascular disease comprising administering to apatient at risk thereof of an effective amount of a compound firstidentified as an ABCA1 modulator using the method as disclosed herein.In preferred embodiments thereof, the disease is coronary artery diseaseor atherosclerosis.

The following examples and methodology were used in effecting thedisclosure herein.

Identification of SNPs

SNPs in the ABC1 gene were identified during the sequencing of 16unrelated probands with low HDL-C^(9,10,14) and of BAC (bacterialartificial chromosome) clones spanning the entire region. By definition,SNPs result from the substitution of one nucleotide with another, whileother polymorphisms can result from the insertion or deletion of one ormore nucleotides²⁰. For simplification, we have used the term SNP torefer to all variants that have been found in our study populations. TheUTR SNPs are numbered from the nucleotide described as position 1²¹,naming the first exon number 1. Nucleotides within the promoter arenumbered according to their position upstream of the transcription startsite, with at −1 as the first nucleotide upstream of that site. Theintronic sites are numbered as their position upstream of the 3′ end ofintron 1, with the most 3′ nucleotide of the intron as position −1.

Subjects

To assess the effects of these SNPs on lipid levels and CAD, we studieda cohort of 804 Dutch men with proven CAD who participated in theRegression Growth Evaluation Statin Study (REGRESS), which haspreviously been described²². The REGRESS and its DNA substudies wereapproved by the institutional review boards and medical ethicscommittees of all participating centres.

For replication studies, the SNPs were screened in a cohort ofindividuals with familial hypercholesterolemia, which was available inthe lab and has previously been described¹⁵ (and Clee et al, manuscriptsubmitted).

Coronary Artery Disease Measurements

Computer-assisted quantitative coronary angiography was carried out atthe start and at the end of the study as previously described²². Themean segment diameter (MSD) measures the average unobstructed diameteralong the vessel, a measure of diffuse atherosclerosis. The minimumobstruction diameter (MOD) represents the unobstructed diameter at thesite of maximal obstruction, reflecting focal atherosclerosis. Largermeasurements of MSD and MOD thus reflect less occlusion of the vessel.The changes in these parameters (delta-MSD and delta-MOD) during the twoyear study, were calculated as the baseline measurement minus thefollow-up measurement. Thus larger values of the delta-MSD and delta-MODreflect increased progression of coronary atherosclerosis. In addition,the incidence of cardiovascular events (death, myocardial infarction,unscheduled coronary angioplasty or bypass surgery (PTCA, CABG), orstroke/transient ischemic attack) during the study was examined.

In the replication cohort, vascular disease was described as any form ofcoronary artery disease (myocardial infarction, CABG, PTCA, anginatreated with medication, angiographic evidence of CAD), cerebrovasculardisease (stroke, transient ischemic attack) or peripheral vasculardisease (individuals with claudication and surgery on carotid orabdominal arteries, not including individuals with bruits only,aneurysms, or evidence only from ultrasound).

SNP Screening

For each variant, we identified a restriction enzyme whose cleavagepattern was altered by the variant for development of an RFLP assay. Ifno suitable enzyme was found, we designed a mismatch primer, whereby asingle nucleotide mismatch was incorporated into the primer, creating arestriction site in combination with either the wildtype or variantallele. The specific conditions of all assays are described in Table 1(SEQ ID NOS: 1-24). All PCR reactions were carried out in the presenceof 1.5 μM MgCl₂ (Life Technologies). Thermocycling parameters were asfollows: 96° C. for 5 minutes; 33 cycles of 96° C. 10 seconds, 30seconds at the annealing temperature specified in Table 1, 1 minute at72° C.; and ended with a final elongation at 72° C. for 10 minutes. Alldigestions were carried out for 2 hours under the conditions specifiedby the manufacturer (New England Biolabs).

Large-scale screening of the variants in intron 1 was performed withTaqMan® based PCR assays^(23,24). Briefly, two fluorogenic hybridizationprobes (one for each allele) are labelled with different fluorescentreporter dyes at their 5′ terminus and a common quencher dye at their 3′terminus. The probes are cleaved by the 5′ nuclease activity of Taqenzyme during PCR amplification, separating the reporter dye from thequencher. The fluorescence of each dye in each reaction was normalizedto the signal from no-DNA controls and compared to known genotypestandards included on each plate.

As a standardised nomenclature for all variants, the allele that wasmore frequent in the REGRESS population was designated A, while thevariant (less frequent) allele was designated B (Table 1 (SEQ ID NOS:1-24)).

Statistics

We compared the baseline characteristics of the individuals in theREGRESS population in the three genotypes (AA, AB, BB) using one-wayanalysis of variance, and the chi-square test, where appropriate. Wealso compared AA versus the combined carrier group (AB+BB) or thehomozygous carriers (BB) using a t-test. P-values unadjusted formultiple comparisons are presented to allow the reader to judge therelative significance of the findings. The cumulative event incidencewas compared using the logrank test and are presented as Kaplan Meiercurves. The change in MOD and MSD and events during the trial weremeasured following randomization to placebo and pravastatin, which wasassessed by chi-square analysis and was equal for all variants exceptthe InsG319, where all 6 BB individuals were randomized to placebo.These parameters were also analyzed in the placebo and pravastatinsubgroups separately, and unless otherwise stated, the results for thetwo treatment groups were comparable, and the combined results arepresented. All lipid levels are reported in mmol/L. All values arereported as mean±standard deviation.

Methods for Identifying SNPs in a Patient Sample.

All means of identifying DNA sequences specific to an individual arecontemplated by this invention.

In general, the detection of single nucleotide polymorphism and singlebase mutation or variation requires a discrimination technique,optionally an amplification reaction and optionally a signal generationsystem. There are numerous techniques available for typing SNPs andallelic variations (for review, see Eberle & Kruglyak Genet Epidemiol2000;19 Suppl 1:S29-35; Kennedy EXS 2000;89:1-10; Kao et al. Ann AcadMed Singapore May 2000;29(3):376-82; Kao et al. Ann Acad Med SingaporeMay 2000;29(3):376-82; Landegren et al., Genome Research, Vol. 8, pp.769-776,1998; Nollau et al, Clin. Chem. 43,1114-1120, 1997 and instandard textbooks, for example ‘Laboratory Protocols for MutationDetection’, Ed, Landegren, Oxford University Press, 1996 and ‘PCR’ 2ndEdition by Newton and Graham, BIOS Scientific Publishers limited, 1997).

Techniques include direct sequencing (Carothers et al., BioTechniques,Vol. 7, pp. 494-499,1989), single-strand conformation polymorphism(SSCP, Orita et al., Proc. Natl. Acad. Sci. USA, Vol. 86, pp.2766-2770,1989), allele-specific amplification (Newton et al., NucleicAcids Research, Vol. 17, pp. 2503-2516,1989), restriction digestion (Dayand Humphries, Analytical Biochemistry, Vol. 222, pp. 389395, 1994),restriction fragment length polymorphism (RFLP) and hybridizationassays. Other methods include high density arrays, mass spectrometry,molecular beacons, peptide nucleic acids, and mismatch cleavage basedassays. These include but are not limited to bacteriophage T4endonuclease VII (U.S. Pat. No. 6,110,684 issued Aug. 29, 2000; U.S.Pat. No. 6,183,958 issued Feb. 6, 2001, U.S. Pat. No. 5,958,692, U.S.Pat. No. 5,851,770, WO 00/18967 Apr. 6, 2000; WO 00/50639 Aug. 31, 2000)WO 00/18967). 5′ nucleases and/or 3′ exonucleases (U.S. Pat. No.5,888,780, WO 98/50403A1, U.S. Pat. No. 5,719,028, WO 00/66607;WO056925) and others such as WO 073766, WO050871, WO 00/66607).

Techniques can also be classified as either target amplification orsignal amplification. Target amplification involves the amplification(i.e., replication) of the target sequence to be detected, resulting ina significant increase in the number of target molecules. Targetamplification strategies include the polymerase chain reaction (PCR),strand displacement amplification (SDA), and nucleic acid sequence basedamplification (NASBA). Signal amplification strategies include theligase chain reaction (LCR), cycling probe technology (CPT), invasivecleavage techniques such as Invader™ technology, Q-Beta replicase (QBR)technology, and the use of “amplification probes” such as “branched DNA”that result in multiple label probes binding to a single targetsequence.

Further assays include, but are not limited to, ligation based assays,cleavage based assays (mismatch and invasive cleavage such as Invader™),and single base extension methods (see WO 92/15712, EP 0 371 437 B1, EP0317 074 B1; Pastinen et al., Genome Res. 7: 606-614 (1997); Syvãnen,Clinica Chimica Acta 226: 225-236 (1994); and WO 91/13075).

The polymerase chain reaction (PCR) is widely used and described, andinvolves the use of primer extension combined with thermal cycling toamplify a target sequence; see U.S. Pat. Nos. 4,683,195 and 4,683,202,and PCR Essential Data, J. W. Wiley & sons, Ed. C. R. Newton, 1995, allof which are incorporated by reference. In addition, there are a numberof variations of PCR which also find use in the invention, including“quantitative competitive PCR” or “QC-PCR”, “arbitrarily primed PCR” or“AP-PCR”, “immuno-PCR”, “Alu-PCR”, “PCR single strand conformationalpolymorphism” or “PCR-SSCP”, allelic PCR (see Newton et al. Nucl. AcidRes. 17: 2503 91989); “reverse transcriptase PCR” or “RT-PCR”, “biotincapture PCR”, “vectorette PCR”. “panhandle PCR”, and “PCR select cDNAsubtraction”, Multiplex PCR amplification of SNP loci with subsequenthybridization to oligonucleotide arrays has been shown to be an accurateand reliable method of simultaneously genotyping at least hundreds ofSNPs; see Wang et al., Science, 280: 1077 (1998); Schafer et al., NatureBiotechnology 16: 33-39 (1998).

Strand displacement amplification (SDA) is generally described in Walkeret al., in Molecular Methods for Virus Detection, Academic Press, Inc.,1995, and U.S. Pat. Nos. 5,455,166 and 5,130,238, all of which arehereby incorporated by reference. Nucleic acid sequence basedamplification (NASBA) is generally described in U.S. Pat. No. 5,409,818and “Profiting from Gene-based Diagnostics”, CTB InternationalPublishing Inc., N.J., 1996, both of which are incorporated by referencein their entirety.

Cycling probe technology (CPT) is a nucleic acid detection system basedon signal or probe amplification rather than target amplification, suchas is done in polymerase chain reactions. Cycling probe technologyrelies on a molar excess of labelled probe that contains a scissilelinkage of RNA. Upon hybridization of the probe to the target, theresulting hybrid contains a portion of RNA: DNA. This area of RNA: DNAduplex is recognized by RNAse H and the RNA is excised, resulting incleavage of the probe. The probe now consists of two smaller sequenceswhich may be released, thus leaving the target intact for repeatedrounds of the reaction. The unreacted probe is removed and the label isthen detected. CPT is generally described in U.S. Pat. Nos. 5,011,769,5,403,711, 5,660,988, and 4,876,187, and PCT published applications WO95/05480, WO 95/1416, and WO 95/00667, all of which are specificallyincorporated herein by reference. Invader™ technology is based onstructure-specific polymerases that cleave nucleic acids in a sitespecific manner. Two probes are used: an “invader” probe and a“signalling” probe, that adjacent hybridize to a target sequence with anon-complementary overlap. The enzyme cleaves at the overlap due to itsrecognition of the “tail”, and releases the “tail” with a label. Thiscan then be detected. The Invader technology is described in U.S. Pat.Nos. 5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669, all ofwhich are hereby incorporated by reference.

The oligonucleotide ligation assay (OLA), sometimes referred to as theligation chain reaction (LCR)), involve the ligation of at least twosmaller probes into a single long probe, using the target sequence asthe template for the ligase. See generally U.S. Pat. Nos. 5,185,243,5,679,524 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182B1; WO 90/01069; WO 89/12696; and WO 89/09835.

“Rolling circle amplification” is based on extension of a circular probethat has hybridized to a target sequence. A polymerase is added thatextends the probe sequence. As the circular probe has no terminus, thepolymerase repeatedly extends the circular probe resulting inconcatamers of the circular probe. As such, the probe is amplified.Rolling-circle amplification is generally described in Baner et al.,(1998) Nuc. Acids Res. 26: 5073-5078; Barany, F. (1991) Proc. Natl.Acad. Sci. USA 88: 189-193; and Lizardi et al., (1998) Nat Genet. 19:225-232, all of which are incorporated by reference in their entirety.

Branched DNA signal amplification (BDNA) relies on the synthesis ofbranched nucleic acids, containing a multiplicity of nucleic acid “arms”that function to increase the amount of label that can be put onto oneprobe. This technology is generally described in U.S. Pat. Nos.5,681,702, 5,597,909, 5,545,730, 635,352, 5,594,118, 5,359,100,5,124,246 and 5,681,697, all of which are hereby incorporated byreference. Similarly, dendrimers of nucleic acids serve to vastlyincrease the amount of label that can be added to a single molecule,using a similar idea but different compositions. This technology is asdescribed in U.S. Pat. No. 5,175,270 and Nilsen et al., J. Theor. Biol.187: 273 (1997), both of which are incorporated herein by reference.

Other methods include mismatch detection techniques using enzymaticcleavage such as resolvase (Variagenics resolvase, bacteriophage T4endonuclease VII, U.S. Pat. No. 6,110,684, issued Aug. 29, 2000; U.S.Pat. No. 6,183,958, issued Feb. 6, 2001, U.S. Pat. No. 5,958,692, U.S.Pat. No. 5,851,770, WO 00/18967 Apr. 6, 2000; WO 00/50639 published Aug.31, 2000) WO 00/18967). The use of 5′ nucleases and/or 3′ exonucleasesfor target dependent reactions using cleavage structures (Third WaveU.S. Pat. No. 5,888,780, WO 98/50403A1, U.S. Pat. No. 5,719,028, Aclara(WO 00/66607; WO056925). Orchid Biosciences (WO 073766, WO050871, WO00/66607).

Screening Patients Having Low HDL-C or High Triglyceride Levels

ABC1 expression, biological activity, and mutational analysis can eachserve as a diagnostic tool for low HDL or higher than normaltriglyceride levels; thus determination of the genetic subtyping of theABC1 gene sequence can be used to subtype low HDL or higher than normaltriglyceride individuals or families to determine whether the low HDL orhigher than normal triglyceride phenotype is related to ABC1 function.This diagnostic process can lead to the tailoring of drug treatmentsaccording to patient genotype, including prediction of side-effects uponadministration of HDL increasing or triglyceride lowering drugs(referred to herein as pharmacogenomics). Pharmacogenomics allows forthe selection of agents (e.g., drugs) for therapeutic or prophylactictreatment of an individual based on the genotype of the individual(e.g., the genotype of the individual is examined to determine theability of the individual to respond to a particular agent).

Agents, or modulators which have a stimulatory or inhibitory effect onABC1 biological activity or gene expression can be administered toindividuals to treat disorders (e.g., cardiovascular disease, low HDLcholesterol, or a higher than normal triglyceride level) associated withaberrant ABC1 activity. In conjunction with such treatment, thepharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) of the individual may be considered. Differences inefficacy of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, the pharmacogenomics of theindividual permits the selection of effective agents (e.g., drugs) forprophylactic or therapeutic treatments based on a consideration of theindividual's genotype. Such pharmacogenomics can further be used todetermine appropriate dosages and therapeutic regimens. Accordingly, theactivity of ABC1 protein, expression of ABC1 nucleic acid, or mutationcontent of ABC1 genes in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons (Eichelbaum, M., Clin. Exp. Pharmacol.Physiol., 23:983-985, 1996; Linder, M. W., Clin. Chem., 43:254-266,1997). In general, two types of pharmacogenetic conditions can bedifferentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). Altered drug action may occur in apatient having a polymorphism (e.g., an single nucleotide polymorphismor SNP) in promoter, intronic, or exonic sequences of ABC1. Thus bydetermining the presence and prevalence of polymorphisms allow forprediction of a patient's response to a particular therapeutic agent. Inparticular, polymorphisms in the promoter region may be critical indetermining the risk of HDL deficiency, higher than normal triglyceridelevel, and CVD.

In addition to the mutations in the ABC1 gene described herein, we havepreviously detected polymorphisms in the human ABC1 gene (PCTPublication WO 00/55318, filed Mar. 15, 2000; U.S. Utility applicationSer. No. 09/526,193, filed Mar. 15, 2000; PCT Publication WO 00/0115676,filed Sep. 1, 2000; or U.S. Utility application Ser. No. 09/654,323,filed Sep. 1, 2000). These polymorphisms are located in promoter,intronic, and exonic sequence of ABC1. Using standard methods, such asdirect sequencing, PCR, SSCP, or any other polymorphism-detectionsystem, one could easily ascertain whether these polymorphisms arepresent in a patient prior to the establishment of a drug treatmentregimen for a patient having low HDL, a higher than normal triglyceridelevel, cardiovascular disease, or any other ABC1-mediated condition. Itis possible that some these polymorphisms are, in fact, weak mutations.Individuals harbouring such mutations may have an increased risk forcardiovascular disease; thus, these polymorphisms may also be useful indiagnostic assays.

Results

During the sequencing of 16 probands with TD or FHA^(9,10,14), we haveidentified 12 SNPs in regions potentially involved in regulatoryfunctions: 2 in the promoter, 4 in the 5′ UTR encoded by exons 1 and 2,and 6 within intron 1 (FIG. 1).

While sequencing, it became apparent that certain pairs of variants werein complete or near complete linkage disequilibrium with each other(FIG. 1, dashed lines). All individuals carrying the InsG319 variant (5heterozygous, 2 homozygous) had the identical genotype at G378C,confirming a previous report of linkage disequilibrium between these twoSNPs²¹. Of the 16 individuals sequenced, three individuals wereheterozygous and 5 were homozygous for both the A-461C and A-362Gvariants; although, 1 individual was homozygous for the A-362G variantbut did not carry the variant at −461. Finally, 2 of the 16 individualswere heterozygous and 5 homozygous for both the G-720A and G-1027Avariants. Thus, for phenotypic analysis, only one variant of each ofthese pairs was analyzed.

Promoter Variants: The G-191C SNP is Associated with Increased and theC-17G with Decreased Coronary Events

We assessed the phenotypic effects of these SNPs in the REGRESS cohort,a well described cohort of Dutch men with proven CAD²². The morefrequent allele of each variant in this cohort was designated A, whilethe less frequent allele was designated B. The carrier and B-allelefrequencies of the SNPs are shown in Table 2.

Individuals with the BB-genotype of the G-191C SNP had triple theincidence of coronary events during the two-year study compared toindividuals with the AA-genotype (BB 33.3% (n=30) vs. AA 11.2% (n=214),p=0.001, Table 3), resulting in an odds ratio of 3.96 in BB individualscompared to AA (95% confidence interval 1.66-9.45, p=0.003), a similarmagnitude as that observed in individuals heterozygous for ABC1mutations¹⁴. This is illustrated in FIG. 2, which shows the cumulativeproportion of individuals who remained event free during the study foreach of the genotypes. The curve is significantly lower for BB'scompared to AA's and AB's (p=0.0005 for AA vs. AB vs. BB, FIG. 2). Infurther support of the relationship of the B allele with an increasedfrequency of CAD, family members of BB-individuals had nearly twice theprevalence of an MI compared to non-carriers (BB 73.3% vs. AA 47.7%,p=0.01). The MSD, which measures diffuse atherosclerosis, was alsoindicative of increased atherosclerosis in BB's, although this did notreach statistical significance (Table 3). However, despite thedifferences in CAD, no significant differences in lipid levels betweenthe genotypes were noted (Table 3).

To further confirm and replicate these findings, we genotyped thisvariant in a cohort of individuals with familial hypercholesterolemiaavailable in the lab¹⁵ (and Clee et al, manuscript submitted). Withinthis cohort, carriers of the G-191C variant had increased vasculardisease compared to non-carriers (14.8% of AA (n=110), 34.3% of AB(n=36), 26.1% of BB (n=48), p=0.03). But, as with the REGRESS cohort, nosignificant differences in TG (AA 1.50±0.75, AB 1.83±0.94, BB 1.44±0.80,p=0.11) or HDL-C (AA 1.29±0.44, AB 1.28±0.38, BB 1.20±0.25, p=0.41)amongst the genotypes were observed.

In contrast to the G-191C, carriers of the C-17G variant had fewerevents during the trial than non-carriers (AB+BB 12.3% (n=351 vs. AA18.2% (n=286, p=0.04, Table 4). Similarly, a smaller percentage ofcarriers had had an MI prior to the start of the study (AB+BB 43.6% vs.AA 52.8%, p=0.02). As with the G-191C, no significant differences inplasma lipid levels were observed between carriers and non-carriers.

This variant was also screened within our replication cohort, andsimilar results were obtained. Homozygous (BB) carriers of this varianthad greater than 3.5-fold fewer events than non-carriers (26.0% of AA(n=80) vs. 7.1% of BB (n=14), p=0.18), although this did not reachstatistical significance. Again, no significant differences in TG (AA1.58±0.91, AB 1.40±0.59 (n=28), BB 1.59±0.55, p=0.61) or HDL-C (AA1.22±0.34, AB 1.30±0.41, BB 1.31±0.55, p=0.56) were observed between thegenotypes.

SNPs in the 5′UTR: The C69T SNP is Associated with Increased and theInsG319 with Decreased Atherosclerosis

Carriers (AB+BB) of the C69T SNP had approximately twice the number ofcoronary events during the trial as non-carriers (AA; p=0.03, Table 5).This difference is illustrated in the event-free survival curve (p=0.03,FIG. 3). Within the placebo group, carriers of the C69T variant hadincreased progression of diffuse atherosclerosis (delta-MSD) compared tonon-carriers (AA vs. AB+BB, p=0.01, Table 5). We observed a similartrend for the progression of focal atherosclerosis in the placebo group(p=0.11, Table 5). No difference in progression of atherosclerosis wasseen for the group treated with pravastatin, or the whole group, whichsuggests that pravastatin may be able to overcome the effects of thisvariant. As with the promoter variants, no differences in mean lipidlevels were observed in carriers of this variant compared tonon-carriers (Table 5).

Increased vascular disease was also observed in carriers of this variantin our replication cohort (18.8% of AA (n=192) vs. 32.2% of AB+BB(n=59), p=0.046; OR=2.05 (95% Cl 1.06-3.96). Once again, no significantdifferences in lipid levels were observed between carriers andnon-carriers of this variant within the replication group (TG: 1.59±0.85vs. 1.62±0.75, p=0.84; HDL: 1.28±0.37 vs. 1.23±0.35, p=0.38; for AA vs.AB+BB, respectively).

In REGRESS, carriers of the C117G SNP had a gene-dose-dependent increaseof TG (AA 1.82±0.79, AB 2.69±0.40, BB 2.77±0.26, p=0.003 for AA vs. BB,Table 4) compared to non-carriers. No other differences in CAD or lipidlevels were observed in C117G carriers. However, this trend was notmaintained within the replication cohort (TG: 1.63±0.86, n=2581.30±0.48, n=9 AA vs. AB+BB, p=0.08), although the number of carriersidentified in this cohort was small. No significant differences in HDL-Cor vascular disease were observed in this cohort (data not shown).

Linkage disequilibrium between the InsG319 and G378C SNPs was confirmedin the REGRESS population, by screening a subset of individuals for bothvariants. The InsG319 and the G378C variants were in almost completelinkage disequilibrium (D′=0.90, p=0.13): of the 59 individuals thatwere screened for both these variants, the genotype of only 1 individualwas discordant (AA for InsG319 and AB for G378C). The remainder (41 AA,15 AB, 2 BB) were identical at both loci. The entire REGRESS cohort wasthus genotyped for only the InsG319.

Carriers of this variant have less focal atherosclerosis (MOD: AA1.76±0.36 vs. AB 1.82±0.34 vs. BB 1.94±0.32, p=0.05, Table 4) and lessdiffuse atherosclerosis (MSD: AA 2.71±0.37 vs. AB 2.84±0.33 vs. BB3.13±0.56, p<0.001) compared to non-carriers. No differences in eventsor mean lipid levels were observed in either the REGRESS or replicationcohort (n=386 AA, 120 AB, 7 BB; data not shown).

Intronic SNPs

Homozygous carriers of the A-1095G SNP had an increased progression offocal atherosclerosis was significantly higher in BB's than in AA's inthe placebo group (delta-MOD: AA 0.13±0.22 (n=216) vs. BB 0.42±1.00(n=5), p=0.01). This trend was not observed in the pravastatin group(delta-MOD: AA 0.07±0.26 (n=222) vs. BB −0.05±0.18 (n=7), p=0.22).Furthermore, the history of CAD in their families was increased comparedto AA-individuals (AA 50.1% vs. BB 83.3%, p=0.02). Consistent with this,trends towards decreased HDL-C (AA 0.93±0.24 vs. BB 0.83±0.20, p=0.15)and increased TG (AA 1.78±0.76 vs. BB 2.18±0.78, p=0.07) were observedin homozygous carriers compared to non-carriers (Table 4). Similartrends were observed in homozygous carriers within the replicationcohort. BB individuals (n=12) had increased vascular disease (50% vs.20.5% in AA's (n=327), p=0.03; OR=3.88, 95% Cl 1.21-12.42), and althoughTG were increased (1.71±0.96 vs. 1.64±0.85) and HDL-C decreased(1.15±0.33 vs. 1.26±0.33), neither finding was statistically significant(p=0.81, 0.29, respectively).

No significant differences in CAD or lipid levels were observed incarriers of the InsCCCT-1163, the G-720A (and G-1027A) or the A-362G(and A-461C)SNPs (Table 5). As observed during sequencing, the G-1027Avariant was in near complete linkage disequilibrium with the G-720Avariant (D′=0.84, p=0.02) in the subset of REGRESS screened for both:all 15 individuals that were BB for G-1027A were also BB for G-720A, andof the 31 individuals that were AB for the G-1027A, 30 individuals werealso AB for G-720A; the other was BB. Linkage disequilibrium between theA-461C and the A-362G variants was also confirmed in REGRESS (D′=0.76,p=0.06): all individuals screened for both these variants (n=52) had thesame genotype for both the variants (n=29 AB, 6 BB).

Partial Linkage Disequilibrium Between SNPs does not Alter thePhenotypic Effects of each SNP

In addition to the near complete linkage disequilibrium alreadydescribed, partial linkage disequilibrium between variants may alsoexist, and thus the phenotype attributed to one variant may be partiallyaccounted for by its association with another functional variant in someof the carriers. We have addressed the issue of potential partiallinkage disequilibrium between the SNPs by examining their pair-wiseassociations (Table 6 and see ref. 15). Of the SNPs that were associatedwith functional effects, 3 pairs were significantly associated with eachother: the C69T with the G-191C, the C17G with the C117G, and theA-1095G with the InsG319. We therefore examined each of these pairs inmore detail.

Nearly half (45%) of the G-191C carriers were also carriers of the C69T.To examine whether the functional effects of the G-191C are independentof the C69T, we examined the G-191C in the subgroup of individuals whowere all non-carriers (i.e. AA) of the C69T (G-191C: AA n=200, AB n=48,BB n=15). Statistical analysis after excluding carriers of the C69Tvariant yielded similar results compared to the analysis without thiscorrection. Coronary events were still increased approximately 3-fold inhomozygous carriers of the G-191C (AA 11%, AB 10.4%, BB 33.3%, p=0.01for AA vs. BB), as was a family history of CAD (AA 49.5%, AB 39.6%, BB73.3%, p=0.08 for AA vs. BB). Furthermore, no differences in plasmalipid levels were unmasked by the exclusion of C69T carriers. Thissuggests that the functional effects attributed to the G-191C variantare not due to effect of the C69T variant.

Unfortunately, as 87% of C69T carriers also have the G-191C variant, toofew carriers of the C69T variant were left (AB+BB n=8) for reasonablestatistical power after exclusion of G-191C carriers. Therefore, it isuncertain whether the effects ascribed to the C69T are due to theG-191C.

A smaller percentage of C-17G carriers were also carriers of the C117G(17%). We performed statistical analysis for the C-17G SNP in thesubgroup of individuals without the C17G variant (C-17G: AA 215, AB 205,BB 28). Similar results were obtained as for the whole group: eventswere reduced (11.6 vs. 18.6%, p=0.04), as were MIs prior to the trial(44.2 vs. 53.0%, p=0.06) for AB+BB compared to AA. Thus, the functionaleffects described for the C-17G SNP are not due to the C117G. Still, nosignificant differences in plasma lipid levels were observed.

Of the carriers of C117G, 82% were also carriers of C-17G. Thus,selection of carriers of C17G who did not have the C-17G variant did notresult in enough carriers to perform statistical analysis. However, ascarriers of the C-17G do not show any differences in plasma TG levels,these trends observed for carriers of the C117G are unlikely to be dueto the co-presence of the C-17G SNP.

As all carriers of the InsG319 were carriers of the A-1095G, we couldnot select a subgroup of InsG319 carriers who did not have the A-1095Gvariant. However, to address if the phenotype of the InsG319 variant isindependent of the A-1095G variant, we performed statistical analysis oncarriers of the InsG319 variant compared to non-carriers, in thesubgroup of individuals who were carriers (AB's and BB's) of the A-1095Gvariant (InsG319: AA n=53 and AB+BB n=87). Keeping carrier status forthe A-1095G constant, there was still significantly less diffuseatherosclerosis (2.86±0.35 vs. 2.70±0.37, p=0.01) and a trend towardsless focal atherosclerosis (1.84±0.35 vs. 1.76±0.33, p=0.18) in carriersof the InsG319 (AB+BB) compared to non-carriers (AA). No significantdifferences in lipid levels were observed, although there were mildtrends towards higher HDL-C (0.93±0.24 vs. 0.88±0.17, p=0.19) and lowerTG (1.66±0.69 vs. 1.84±0.85, p=0.17) between these groups. This suggeststhat atherosclerosis is reduced in InsG319 carriers, independent of theA-1095G variant.

Sixty-three percent of the carriers of A-1095G were also carriers ofInsG319. Following exclusion of the InsG319 carriers, no A-1095G BBindividuals (in whom the phenotype was observed) remained for analysis.However, as the InsG319 was not associated with alterations in plasmalipid levels and was associated with an opposite effect on vasculardisease, the effects of the A-1095G are unlikely to be due to theInsG319.

Here we present an analysis of 12 non-coding SNPs in the promoter,intron 1 and 5′UTR of the ABC1 gene. We report that several of thesecommon variants are associated with altered severity ofarteriosclerosis, without any observed changes in plasma lipid levels(summarized in Table 7).

The G-191C SNP, independent of the C69T SNP with which it is in partiallinkage disequilibrium, was associated with an approximately 3-foldincrease in coronary events, resulting in an odds ratio similar to thatof individuals heterozygous for mutations in ABC1. In further support ofthis was a significantly increased family history of CAD. Increasedvascular disease was also observed in our replication cohort, but inneither cohort were significant differences in plasma lipid levelsobserved.

Similarly, the C69T SNP was also associated with increased coronaryevents and increased atherosclerotic progression, again with nodifferences in plasma lipid levels. These findings were also observed inour replication cohort. Although this variant is in partial linkagedisequilibrium with the G-191C SNP that had similar effects, the C69Tvariant was associated with increased events in both homozygous andheterozygous carriers of the variant, the majority of whom wereheterozygous for the G-191C, whereas the G-191C SNP only showedincreased events in homozygous carriers in REGRESS. Thus, the effects ofthe C69T are not likely to be due entirely to the G-191C SNP.

In contrast, both the C-17G and InsG319 SNPs were associated withreduced arteriosclerosis. The C-17G SNP was associated with a reductionin coronary events both during and prior to the REGRESS study, and witha 3.5-fold reduction in vascular disease events in the replicationcohort. The InsG319 SNP was associated with reduced focal and diffusearteriosclerosis. These effects were independent of other SNPs found inpartial linkage disequilibrium with these variants. As with the G-191Cand C69T SNPs, no significant differences in plasma lipid levels wereobserved in carriers of either variant.

Thus, several ABC1 regulatory variants were associated with an alteredrisk of CAD but without corresponding differences in lipid levels. Thesefindings suggest that decreases or increases in RCT activity may changethe net flux of cholesterol from the vessel wall towards the liver,without altering plasma lipid levels. Thus, ABC1 variation may directlyinfluence the atherosclerotic process without altering plasma lipidlevels. One explanation for these findings might be that only largerchanges in efflux result in measurable changes in plasma lipid levels,whereas smaller changes might still directly impact cholesterolaccumulation within the vessel wall. Indeed, cholesterol efflux ishighly correlated to vessel wall intima-media thickness (van Dam et al,manuscript in preparation). Alternatively, these variants may influenceABC1 regulation in certain tissues (e.g. macrophages) or under someenvironmental stimuli (e.g. in response to cholesterol loading or otheratherogenic stimuli) but not others, and thus may directly influenceABC1 activity within the vessel wall but not elsewhere. Furthermore,lesion macrophages likely constitute a small percentage of total bodycells eliminating excess cholesterol and contributing to plasma HDL-Clevels, and thus changes in macrophage ABC1 activity may not directlyresult in changes in plasma HDL-C levels. Therefore alterations in ABC1regulation may impact cholesterol accumulation specifically in thevessel wall without changing plasma lipid levels.

The A-1095G SNP was also associated with more progression of focalarteriosclerosis and more CAD in family members of the REGRESSparticipants, and with increased vascular disease events in thereplication cohort. In both cohorts there were mild trends towardsdecreased HDL-C and increased TG in carriers, however in neither casewere they significant. Therefore, this variant may either exert a verymild effect, or no effect on plasma lipid levels, again suggesting thatABC1 regulatory variants may have a significant influence on CAD withoutobvious changes in plasma lipid levels.

The C117G SNP was the only SNP directly associated with altered plasmalipid levels, being associated with increased TG levels in the REGRESScohort. However, these findings were not observed within the smallnumber of carriers in the replication cohort. Thus it is uncertainwhether this variant truly influences plasma TG levels. Analysis inadditional cohorts will be required to ascertain whether this SNPaffects plasma lipid levels.

The precise mechanism behind the regulatory function of these SNPs willrequire further analysis. It is possible that the nucleotidesubstitutions directly alter transcription factor binding sites, thusinfluencing the transcription of ABC1. It is also possible thattranscription occurs at a normal rate from the two alleles, but thatmRNA stability is altered or that the two allelic forms of the mRNApossess different abilities to initiate translation, perhaps as a resultof differences in secondary structure²⁵. The SNPs may also influencesplice sites, their recognition, or splicing enhancers²⁶. SNPs withinnon-coding regions have previously been shown to have such functionaleffects^(16-18,26-28). To understand the mechanism by which these SNPsinfluence ABC1 expression, they will need to be re-created in vitro andtheir functionality at basal levels and in response to variousregulatory stimuli in various cell types assessed.

This is the first report describing the in vivo effects of regulatoryvariants within ABC1, and demonstrating that these variants havesignificant effects on the population risk of CAD. Here, we have shownthat several common SNPs in non-coding regions of the ABC1 gene areassociated with an altered risk of CAD in the absence of detectablechanges in plasma lipids.

These findings suggest that proper regulation of ABC1 is critical forRCT and thus prevention of atherosclerotic vascular disease. Asdescribed previously, identification of polymorphisms in the non-codingregion of ABCA1 can provide valuable information for predictivediagnosis of cardiovascular and other disorders and diseases. Based onthe disclosure herein, those skilled in the art can develop nucleic acidsequencing/analysis compositions methods and kits that are suitable fordiagnosis of these diseases. Any method of determining the target genesequence can be used in the method of this invention, including fulllength or partial gene sequencing, probe based assays, RFLP and allother techniques known to those in the art.

Furthermore, sequence analysis of the non-coding region of ABCA1 canalso be used to predict drug responsiveness, susceptibility toside-effects of drugs, and, importantly, it is useful for designingclinical trials, as generally encompassed by the concept ofpharmacogenetics. The polymorphisms or mutations disclosed herein can becorrelated to a patient response database in order to generate aprognostic database for aiding selection of an appropriate therapeuticregime for a patient. Single nucleotide polymorphisms (SNPs) in ABCA1are related to drug responsiveness, drug side effects, and areimplicated in diseases and disorders disclosed in this invention.

Clinical trials for therapeutic agents for treatment of cardiovascularand other diseases and disorders can be simplified and made moreaccurate by performing sequence analysis of the non-coding region ofABCA1 as identified herein. In one embodiment, patients enrolled in aclinical trial for a new therapeutic agent give a tissue sample, and thenucleic acid sequence of the non-coding region of ABCA1 is determined.Patients are categorized by their particular genetic variant and theirresponse to the therapeutic agent. A correlation between drugresponsiveness and genetic variant may be determined. This correlationthen becomes an important tool for physicians who prescribe the drug;all patients who are indicated for the drug are first typed for thegenetic variant to ensure that they will have the desired clinicaloutcome.

Alternatively, clinical trial design may be improved by pre-selectingpatients who are likely to have positive outcomes to a therapeutic agentbased on their having preferred genetic variants of the therapeutictargets disclosed herein. All potential patients are first sequenced atthe relevant target gene, and only those that have the preferred variantare enrolled in the trial. This technique will greatly reduce the numberof patients that are required in a clinical trial to determine efficacyof the therapeutic agent.

In accordance with the foregoing, the present invention alsocontemplates a method for identifying a therapeutic agent foradministration to a patient in need thereof, comprising comparing anucleotide sequence of a non-coding region of an ABCA1 gene of saidpatient to a database (such as where the database comprises ABCA1nucleotide sequences comprising the polymorphic sequences disclosed inTable 1) that correlates nucleic acid sequences of ABCA1 genes with theeffectiveness of therapeutic agents in beneficially regulating lipidlevels in a patient, thereby identifying a therapeutic agent foradministration to said patient.

Further, the present invention provides a method for identifying acandidate for enrolment in a program of clinical trials of a potentialtherapeutic agent, comprising comparing a nucleotide sequence of anon-coding region of an ABCA1 gene of said candidate to a database thatcorrelates nucleic acid sequences of ABCA1 genes with the effectivenessof therapeutic agents in beneficially regulating lipid levels in apatient, thereby identifying a candidate for enrolment in a program ofclinical trials, again especially where the database comprises ABCA1nucleotide sequences comprising the polymorphic sequences disclosed inTable 1.

The present invention also relates to a process that comprises a methodfor producing a product comprising identifying an agent according to oneof the disclosed processes for identifying such an agent (i.e., thetherapeutic agents identified according to the assay proceduresdisclosed herein) wherein said product is the data collected withrespect to said agent as a result of said identification process, orassay, and wherein said data is sufficient to convey the chemicalcharacter and/or structure and/or properties of said agent. For example,the present invention specifically contemplates a situation whereby auser of an assay of the invention may use the assay to screen forcompounds having the desired enzyme modulating activity and, havingidentified the compound, then conveys that information (i.e.,information as to structure, dosage, etc) to another user who thenutilizes the information to reproduce the agent and administer it fortherapeutic or research purposes according to the invention. Forexample, the user of the assay (user 1) may screen a number of testcompounds without knowing the structure or identity of the compounds(such as where a number of code numbers are used the first user issimply given samples labeled with said code numbers) and, afterperforming the screening process, using one or more assay processes ofthe present invention, then imparts to a second user (user 2), verballyor in writing or some equivalent fashion, sufficient information toidentify the compounds having a particular modulating activity (forexample, the code number with the corresponding results). Thistransmission of information from user 1 to user 2 is specificallycontemplated by the present invention.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication wasspecifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations following, in general, the principles of theinvention and including such departures from the present disclosurewithin known or customary practice within the art to which the inventionpertains and may be applied to the essential features hereinabove setforth. TABLE 1 Methods for screening non-coding ABCA1 SNPs Products(bp): Forward oligo (5′-->3′)^(a) “A” allele Annealing wt “A” alleleVariant Reverse oligo (5′-->3′)^(a) “B” allele temp. (C.) Enzyme variant“B” allele G-191C CAGCGCTTCCCGCGCGTCTTAG G 60 HgaI 287, 55, 3 (SEQ IDNO: 1) CCACTCACTCTCGTCCGCAATTAC C 342, 3 (SEQ ID NO: 2) C-17GCTGCTGAGTGACTGAACTACATAAACAGAGGCCGGGTA C 60 Rsa I 161 (SEQ ID NO: 3)CCACTCACTCTCGTCCGCAATTAC G 124, 37 (SEQ ID NO: 4) C69TCAGCGCTTCCCGCGCGTCTTAG C 60 BsmAI 345 (SEQ ID NO: 5)CCACTCACTCTCGTCCGCAATTAC T 310, 35 (SEQ ID NO: 6) C117GCTGGCTTTCTGCTGAGTGAC C 60 Eco 0109 284, 175 (SEQ ID NO: 7) IGATCAAAGTCCCCGAAACC G 459 (SEQ ID NO: 8) A-362GACTCAGTTGTATAACCCACTGAAAATTAGT A 55 Mbo II 224, 26 (SEQ ID NO: 9)TTCTATAGATGTTATCATCTGGG G 134, 90, 26 (SEQ ID NO: 10) A-461CACTCAGTTGTATAACCCACTGAAAATGAGT A 55 Hinf I 150, 100 (SEQ ID NO: 11)TTCTATAGATGTTATCATCTGGG C 123, 100, 27 (SEQ ID NO: 12) G-720ATCATCTAAGGCACGTTGTGG G 60 Hpa II 450 (SEQ ID NO: 13)CCTCAAGCCTGGAGTGACTT A 306, 144 (SEQ ID NO: 14) G-1027AATGGCAAACAGTCCTCCAAG G 60 Nco I 170, 41 (SEQ ID NO: 15)ACCCTAGCGCTGTGTCTCTG A 105, 65, 41 (SEQ ID NO: 16) A-1095GATGGCAAACAGTCCTCCAAG A 60 MspA1 I 211 (SEQ ID NO: 17)ACCCTAGCGCTGTGTCTCTG G 172, 39 (SEQ ID NO: 18) TGTGTGTCCTCCCTTCCATT noins 60 Mnl I 144, 28, 11, 4 (SEQ ID NO: 19) insCCCT-1163CTTGGAGGACTGTTTGCCAT ins 100, 48, 28, (SEQ ID NO: 20) CCCT 11, 4 insG319CCCCTCCTGCTTTATCTTTCAGTTAATGACCAGCCCCG no ins 55 Sma I 246 (SEQ ID NO:21) ATCCCCAACTCAAAACCACA ins G 210, 37 (SEQ ID NO: 22) G378CGCCGCTGCCTTCCAGGGCTCCCGAGCCACACGCTGCG G 55 Aci I 108, 41, 33, 5 (SEQ IDNO: 23) ATCCCCAACTCAAAACCACA C 141, 41, 5 (SEQ ID NO: 24)

TABLE 2 Frequencies of ABCA1 SNPs in the REGRESS Population. Nucleotide“B” Frequency Change allele carrier (%) B-allele N^(a) Promoter G-191C C35.9 0.225 668 C-17G G 55.1 0.323 1274 5′ UTR C69T T 20.4 0.138 812C117G G 11.9 0.065 1096 Ins G319 Ins 16.2 0.085 1396 Intron 1 InsCCCT-1163 Ins 2.1 0.011 1232 A-1095G G 28.2 0.151 1220 G-720A A 68.80.448 910 A-362G G 65.5 0.445 1060^(a)N refers to the number of alleles screened.

TABLE 3 G-191C carriers compared to non-carriers P-value AA vs. AA vs.AA vs. AB vs. AA AB BB BB AB + BB BB n 214 90 30 age 56.6 ± 8.0  59.8 ±8.8  55.3 ± 8.2  0.41 0.07 0.18 Total Cholesterol 5.98 ± 0.87 6.17 ±0.90 6.12 ± 1.10 0.43 0.08 0.22 LDL Cholesterol 4.24 ± 0.80 4.45 ± 0.824.29 ± 0.91 0.75 0.07 0.14 HDL Cholesterol 0.93 ± 0.23 0.92 ± 0.22 0.94± 0.29 0.83 0.90 0.89 Triglycerides 1.79 ± 0.73 1.78 ± 0.74 1.95 ± 0.770.27 0.73 0.51 MSD 2.71 ± 0.39 2.73 ± 0.40 2.65 ± 0.34 0.19 0.96 0.69MOD 1.74 ± 0.35 1.75 ± 0.33 1.76 ± 0.30 0.83 0.96 events % (n) 11.2(24)  11.1 (10) 33.3 (10) 0.001 0.39 0.003 family history CAD % (n) 47.7(102) 43.1 (31) 73.3 (22) 0.01 0.01 0.02

TABLE 4 Lipid Levels and CAD in Carriers of ABCA1 SNPs. P-values AA vs.AA vs. AB AA AB BB AA vs. BB AB + BB vs. BB C-17G n 286 290 61 HDL 0.92± 0.22 0.93 ± 0.23 0.88 ± 0.21 0.19 0.91 0.33 TG 1.85 ± 0.75 1.77 ± 0.811.98 ± 0.82 0.23 0.52 0.14 MOD 1.77 ± 0.37 1.78 ± 0.35 1.72 ± 0.35 0.330.86 0.55 MSD 2.75 ± 0.39 2.74 ± 0.38 2.65 ± 0.39 0.07 0.48 0.22 events% (n) 18.2 (52) 12.4 (36) 11.5 (7)  0.21 0.04 0.11 C117G n 487  59 6 HDL0.92 ± 0.23 0.96 ± 0.25 0.95 ± 0.17 0.32 0.25 0.51 TG 1.82 ± 0.79 2.69 ±0.40 2.77 ± 0.26 <0.0001 0.45 0.67 MOD 1.75 ± 0.36 1.81 ± 0.40 1.70 ±0.15 0.28 0.32 0.48 MSD 2.74 ± 0.38 2.69 ± 0.40 2.77 ± 0.26 0.55 0.450.67 events % (n) 14.8 (72) 8.5 (5) 33.3 (2)  0.21 0.5 0.19 InsG319 n581 107 6 HDL 0.92 ± 0.23 0.93 ± 0.23 0.89 ± 0.30 0.75 0.88 0.76 TG 1.80± 0.77 1.68 ± 0.66 1.89 ± 0.84 0.78 0.29 0.16 MOD 1.76 ± 0.36 1.82 ±0.34 1.94 ± 0.32 0.22 0.11 0.05 MSD 2.71 ± 0.37 2.84 ± 0.33 3.13 ± 0.560.01 <0.001 <0.001 events % (n) 15.5 (90) 13.1 (14) 16.7 (1)  0.94 0.810.55 InsCCCT-1163 n 603  12 1 HDL 0.93 ± 0.23 0.85 ± 0.18 0.82 0.63 0.210.46 TG 1.76 ± 0.76 1.83 ± 0.81 1.67 0.90 0.81 0.95 MOD 1.76 ± 0.36 1.82± 0.31 1.76 1.00 0.58 0.85 MSD 2.74 ± 0.37 2.83 ± 0.38 2.34 0.28 0.570.37 events % (n) 14.9 (90) 8.3 (1)  100 (1)  0.02 0.96 0.05 A-1095G n438 160 12 HDL 0.93 ± 0.24 0.93 ± 0.22 0.83 ± 0.20 0.15 0.53 0.28 TG1.78 ± 0.76 1.74 ± 0.75 2.18 ± 0.78 0.07 0.88 0.15 MOD 1.76 ± 0.37 1.79± 0.34 1.79 ± 0.41 0.78 0.28 0.56 MSD 2.71 ± 0.38 2.76 ± 0.35 2.98 ±0.57 0.02 0.05 0.03 events % (n) 13.7 (60) 14.4 (23) 16.7 (2)  0.77 0.80.94 G-720A n 142 218 95 HDL 0.89 ± 0.20 0.92 ± 0.22 0.91 ± 0.23 0.480.31 0.52 TG 1.87 ± 0.77 1.86 ± 0.77 1.84 ± 0.80 0.77 0.78 0.96 MOD 1.76± 0.35 1.75 ± 0.37 1.70 ± 0.33 0.19 0.46 0.43 MSD 2.72 ± 0.38 2.72 ±0.35 2.69 ± 0.38 0.55 0.85 0.77 events % (n) 12.7 (18) 14.2 (31) 12.6(12) 0.99 0.88 0.89 A-362G n 182 221 124 HDL 0.94 ± 0.23 0.94 ± 0.240.93 ± 0.2  0.82 0.91 0.97 TG 1.86 ± 0.82 1.72 ± 0.77 1.86 ± 0.76 0.940.22 0.11 MOD 1.75 ± 0.35 1.76 ± 0.35 1.74 ± 0.35 0.98 0.80 0.90 MSD2.69 ± 0.40 2.74 ± 0.37 2.73 ± 0.37 0.38 0.21 0.45 events % (n) 16.5(30) 13.6 (30) 11.3 (14) 0.20 0.24 0.42

TABLE 5 C69T Carriers Compared to Non-Carriers P-value AA vs. AA vs. ABvs. AA AB BB AA vs. BB AB + BB BB n 323 54 29 age 56.1 ± 8.2  55.0 ±8.0  55.7 ± 6.6 0.80 0.40 0.66 Total Cholesterol 6.01 ± 0.87 6.15 ± 0.88 6.19 ± 1.04 0.29 0.17 0.38 LDL Cholesterol 4.28 ± 0.79 4.43 ± 0.78 4.34 ± 0.96 0.70 0.23 0.43 HDL Cholesterol 0.91 ± 0.22 0.91 ± 0.19 0.95 ± 0.30 0.36 0.79 0.70 Triglycerides 1.82 ± 0.75 1.80 ± 0.76  2.00± 0.80 0.22 0.61 0.44 MSD 2.72 ± 0.39 2.68 ± 0.37  2.70 ± 0.33 0.79 0.480.76 MOD 1.74 ± 0.34 1.70 ± 0.36  1.85 ± 0.29 0.09 0.86 0.16 events %(n)12.4 (40) 20.4 (11) 24.1 (7) 0.07 0.03 0.09 Placebo n 159 26 17delta-MSD 0.10 ± 0.21 0.21 ± 0.29  0.22 ± 0.21 0.03 0.01 0.02 delta MOD0.13 ± 0.27 0.19 ± 0.31  0.25 ± 0.26 0.08 0.11 0.25 events % (n) 16.4(26) 26.9 (7)  35.3 (6) 0.05 0.04 0.11 Pravastatin n  64 28 12 delta-MSD0.08 ± 0.19 0.05 ± 0.13 −0.02 ± 0.10 0.08 0.16 0.23 delta-MOD 0.07 ±0.21 0.27 ± 0.97 −0.05 ± 0.15 0.06 0.28 0.11 events % (n)  8.5 (14) 14.3(4)   8.3 (1) 0.28 0.23 0.51

TABLE 6 P-Values for Pairwise Associations of the ABCA1 SNPs in REGRESSinsCCCT- G-191C C-17G C69T C117G 1163 A-1095G G-720A A-362G insG319G-191C — C-17G NA — C69T <0.0001 NA — C117G NA <0.0001 NA — insCCCT- NANA NA NA — 1163 A-1095G NA NA NA NA <0.0001 — G-720A NA NA NA NA NA NA —A-362G NA NA NA NA NA <0.0001 NA — insG319 NA NA NA NA 0.002 <0.0001 NA<0.0001 —NA = not significantly associated

TABLE 7 Associations of ABCA1 SNPs with altered lipid levels and CAD inREGRESS Variant p-value Variant p-value More coronary events Lesscoronary events G-191C 0.001 C-17G 0.04 C69T 0.03 Increased familyhistory of CAD Less MI prior to study G-191C 0.01 C-17G 0.02 A-1095G0.02 Increased progression of atherosclerosis Less atherosclerosis C69T0.01 InsG319 <0.001 A-1095G (focal 0.01 atherosclerosis) Increased TGC117G 0.003

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1. A method for determining propensity toward developing acardiovascular disease in a patient at risk of developing said diseasecomprising determining the presence in an ABCA1 gene of said patient ofa polymorphism in the DNA sequence of said gene wherein saidpolymorphism is present in a non-coding region of said gene.
 2. Themethod of claim 1 wherein said polymorphism is present in the promoterregion of said gene.
 3. The method of claim 1 wherein said polymorphismis present in an intronic region of said gene.
 4. The method of claim 1wherein said disease is coronary artery disease.
 5. The method of claim1 wherein said disease involves increased triglyceride levels in theplasma of said patient.
 6. The method of claim 1 wherein said diseaseinvolves decreased high density lipoprotein (HDL-C) levels in the plasmaof said patient.
 7. The method of claim 1 wherein said disease involveselevated cholesterol levels in the plasma of said patient.
 8. The methodof claim 1 wherein said disease involves decreased lipid transport inthe cells of said patient.
 9. The method of claim 1 wherein saidpolymorphism is a single nucleotide polymorphism.
 10. The method ofclaim 9 wherein said polymorphism is a polymorphism shown in Table 1.11. A method for identifying a modulator of ABCA1 polynucleotideexpression comprising: (a) contacting a compound with a polynucleotidethat encodes ABCA1 polypeptide, which polynucleotide comprises apolymorphism in a non-coding region of said polynucleotide, underconditions promoting said contacting and promoting expression of ABCA1polypeptide by said polynucleotide; (b) determining the activity of saidpolynucleotide in expressing said ABCA1 polypeptide after saidcontacting wherein a difference in the expression of said polynucleotiderelative to when said compound and said polynucleotide are not contactedindicates polynucleotide modulating activity, thereby identifying amodulator of ABCA1 polynucleotide expression.
 12. The method of claim 11wherein said ABCA1 polynucleotide is present in a cell.
 13. The methodof claim 11 wherein said difference in expression in step (b) is anincrease in expression.
 14. The method of claim 11 wherein saidpolymorphism is present in an intronic region of said polynucleotide.15. The method of claim 11 wherein said polymorphism occurs in apromoter region of said polynucleotide.
 16. The method of claim 11wherein said polymorphism is a single nucleotide polymorphism (SNP). 17The method of claim 14 wherein said SNP is a member selected from theSNPs shown in Table
 1. 18. The method of claim 11 wherein saidpolymorphism has the effect of decreasing the activity of saidpolynucleotide.
 19. A method of identifying an agent that modulatesplasma lipid levels comprising administering to an animal an effectiveamount of a compound first identified as an ABCA1 modulator using themethod of claim
 11. 20. The method of claim 19 wherein said compound hasthe effect of reducing plasma triglyceride levels.
 21. The method ofclaim 19 wherein said compound has the effect of reducing plasmacholesterol levels.
 22. The method of claim 19 wherein said compound hasthe effect of increasing plasma HDL-C levels.
 23. A method of treating apatient for cardiovascular disease comprising administering to a patientafflicted therewith of an effective amount of a compound firstidentified as an ABCA1 modulator using the method of claim 11 or
 19. 24.The method of claim 23 wherein said disease is coronary artery disease.25. The method of claim 23 wherein said disease is atherosclerosis. 26.A method of protecting a patient against developing cardiovasculardisease comprising administering to a patient at risk thereof of aneffective amount of a compound first identified as an ABCA1 modulatorusing the method of claim 11 or
 19. 27. The method of claim 26 whereinsaid disease is coronary artery disease.
 28. The method of claim 26wherein said disease is atherosclerosis.
 29. A method for identifying atherapeutic agent for administration to a patient in need thereof,comprising comparing a nucleotide sequence of a non-coding region of anABCA1 gene of said patient to a database that correlates nucleic acidsequences of ABCA1 genes with the effectiveness of therapeutic agents inbeneficially regulating lipid levels in a patient, thereby identifying atherapeutic agent for administration to said patient.
 30. The method ofclaim 29 wherein said database comprises ABCA1 nucleotide sequencescomprising the polymorphic sequences disclosed in Table
 1. 31. A methodfor identifying a candidate for enrolment in a program of clinicaltrials of a potential therapeutic agent, comprising comparing anucleotide sequence of a non-coding region of an ABCA1 gene of saidcandidate to a database that correlates nucleic acid sequences of ABCA1genes with the effectiveness of therapeutic agents in beneficiallyregulating lipid levels in a patient, thereby identifying a candidatefor enrolment in a program of clinical trials.
 32. The method of claim31 wherein said database comprises ABCA1 nucleotide sequences comprisingthe polymorphic sequences disclosed in Table
 1. 33. A method forproducing a product comprising identifying an agent according to theprocess of claim 11 or 19 wherein said product is the data collectedwith respect to said agent as a result of said process and wherein saiddata is sufficient to convey the chemical structure and/or properties ofsaid agent.